Influenza is caused by an RNA virus of the orthomyxoviridae family. There are three types of these viruses and they cause three different types of influenza: type A, B and C. Influenza virus type A viruses infect mammals (humans, pigs, ferrets, horses) and birds. This is very important to mankind, as this is the type of virus that has caused worldwide pandemics. Influenza virus type B (also known simply as influenza B) infects only humans. It occasionally causes local outbreaks of flu. Influenza C viruses also infect only humans. They infect most people when they are young and rarely causes serious illness.
Vaccination provides protection against disease caused by a like agent by inducing a subject to mount a defense prior to infection. Conventionally, this has been accomplished through the use of live attenuated or whole inactivated forms of the infectious agents as immunogens. To avoid the danger of using the whole virus (such as killed or attenuated viruses) as a vaccine, recombinant viral proteins, for example subunits, have been pursued as vaccines. Both peptide and subunit vaccines are subject to a number of potential limitations. Subunit vaccines may exhibit poor immunogenicity, owing to incorrect folding or poor antigen presentation. A major problem is the difficulty of ensuring that the conformation of the engineered proteins mimics that of the antigens in their natural environment. Suitable adjuvants and, in the case of peptides, carrier proteins, must be used to boost the immune response. In addition these vaccines elicit primarily humoral responses, and thus may fail to evoke effective immunity. Subunit vaccines are often ineffective for diseases in which whole inactivated virus can be demonstrated to provide protection.
Virus-like particles (VLPs) are potential candidates for inclusion in immunogenic compositions. VLPs closely resemble mature virions, but they do not contain viral genomic material. Therefore, VLPs are nonreplicative in nature, which make them safe for administration as a vaccine. In addition, VLPs can be engineered to express viral glycoproteins on the surface of the VLP, which is their most native physiological configuration. Moreover, since VLPs resemble intact virions and are multivalent particulate structures, VLPs may be more effective in inducing neutralizing antibodies to the glycoprotein than soluble envelope protein antigens.
VLPs have been produced in plants (WO2009/009876; WO 2009/076778; WO 2010/003225; WO 2010/003235; WO 2011/03522; WO 2010/148511; which are incorporated herein by reference), and in insect and mammalian systems (Noad, R. and Roy, P., 2003, Trends Microbiol 11: 438-44; Neumann et al., 2000, J. Virol., 74, 547-551). Latham and Galarza (2001, J. Virol., 75, 6154-6165) reported the formation of influenza VLPs in insect cells infected with recombinant baculovirus co-expressing hemagglutinin (HA), neuramindase (NA), M1, and M2 genes. This study demonstrated that influenza virion proteins self-assemble upon co-expression in eukaryotic cells and that the M1 matrix protein was required for VLP production. Gomez-Puertas et al., (1999, J. Gen. Virol, 80, 1635-1645) showed that overexpression of M2 completely blocked CAT RNA transmission to MDCK cultures.
The spike glycoprotein hemagglutinin (HA) of influenza viruses is of great importance for the uptake of virus particles by the host cell. It is responsible for their attachment to sialic acid-containing cellular receptors, and it is involved in virus penetration through fusion of the virus envelope with cellular membranes. Fusion activity and consequently virus infectivity depend on cleavage of the HA precursor molecule, HA0, into the disulfide-linked polypeptide chains, HA1 and HA2. Cleavage a subsequent pH-dependent conformational change result in the exposure and relocation of a highly conserved hydrophobic peptide at the amino terminus of the transmembrane polypeptide HA2, which mediates membrane fusion.
HA is synthesised as a precursor protein HA0, which undergoes proteolytic processing into two subunits (HA1 and HA2) linked together by a disulfide bridge. Two structural features are thought to be involved in HA cleavability: in HAs of restricted cleavability, the linker usually consists of a single arginine, whereas HAs cleavable in a broad range of different cell types have an insertion of a series of multiple basic residues in this position with the main enzyme recognition motif Arg-X-Lys/Arg-Arg, whereby X is a nonbasic amino acid. HAs with a multiple basic cleavage site are cleaved on the exocytic transport route before they reach the budding site on the cell surface, in contrast to HAs with a monobasic linker that are activated on virus particles either in the extracellular space or, as shown for the WSN strain, at the stage of virus entry. A second determinant of HA cleavage appears to be a carbohydrate side chain that is present in close vicinity of the cleavage site and interferes with protease accessibility. Loss of this carbohydrate resulted in enhanced HA cleavability and viral pathogenicity.
Mammalian and apathogenic avian influenza virus strains cause anatomically localized infections as a result of the restricted range of cells secreting a protease that can cleave the HA0 precursor extracellularly (Chen J, et. al. 1998, Cell. Vol 95:409-417). The proteases responsible for cleavage of HA0 in influenza infections of humans, are secreted by cells of the respiratory tract, or by coinfecting bacteria or mycoplasma, or they may be produced in inflammatory responses to infections. A major protease candidate is the tryptase Clara, which is produced by Clara cells of the bronchiolar epithelium, and has limited tissue distribution (upper respiratory tract). The protease is specific for the monobasic sequence Q/E-X-R found at the cleavage site of the H1, H2, H3, and H6. HA from H9 and B strains show a slightly different monobasic cleavage site with SSR and KER sequence respectively. No protease has been identified for the majority of influenza viruses that cause enteric and respiratory infection seen in aquatic birds. Most cell lines do not support multi-cycle replication unless exogenous protease (usually trypsin) is added.
In highly pathogenic avian strains, however, HA0 are cleaved by a family of more widespread intracellular proteases, resulting in systemic flu infections. This difference in pathogenicity correlates with structural differences at the HA0 cleavage site. Pathogenic strains have inserts of polybasic amino acids within, or next to, the monobasic site. Cleavage in this case occurs intracellularly and the proteases involved have been identified as furin, and other subtilisin-like enzymes, found in the Golgi and involved in the post-translational processing of hormone and growth factor precursors. The furin recognition sequence R-X-R/K-R is a frequent insertion amino acid at the HA0 cleavage sites of H5 and H7. The wide tissue distribution of the enzyme, and the efficiency of intracellular cleavage, contribute to the wide-spread and virulent systemic infection caused by these viruses.
The HA cleavage site is a target for virus attenuation, since activation cleavage of the HA0 precursor into the HA1 and HA2 fragments by host proteases is a step in the replication cycle of all influenza A and B virus strains. Only the cleaved HA can undergo a conformational change in the acidic milieu of the endosome after receptor-mediated endocytosis to expose the hydrophobic N terminus of the HA2 fragment for mediating fusion between endosomal and virion membranes.
Horimoto T, et. al. (2006, Vaccine, Vol 24: 3669-3676) describes the abolition of the polybasic cleavage site of H5 (RERRRKKR↓G) in H5. Selected mutants were submitted to immunogenicity study in mice, including a mutant with a deletion of the 4 first charged amino acids (RERR) and a modification to inactivate the polybasic cleavage site (RKKR with TETR). Abolition of the cleavage site did not affect the immunogenic properties of the mutant H5. Abolition the polybasic site (GERRRKKR↓G replaced by RETR) to produce mutant NIBSC 05/240 NIBSC influenza reference virus NIBG-23, has also been reported. Hoffman et. al. (2002, 2002, Vaccine, Vol 20:3165-3170) replaced the polybasic cleavage site of a H5 HA with the monobasic site of H6 in order to boost the expression in eggs. The first 4 residues were deleted and replaced the four last amino acids of the polybasic site by IETR (replacement of RERRRKKR↓G with IETR↓G). This mutant H5 showed a high expression level, potential proteolysis and conformational change at low pH required for viral replication and production in the host cell, immunogenicity data were not reported. These studies show that modification of the cleavage site can be employed to diminishes the virulence of the viral particle (in cases where the true viruses is replicated), allowing the virus to replicate without killing the host egg. Without such mutations, viruses kill the egg before reaching high titers.
WO2013043067 by Sirko et al. describe a DNA vaccine for chicken which contains the cDNA encoding a modified H5 haemagglutinin (HA) protein wherein the proteolytic cleavage site between HA subunits is deleted. Sirko et al. state that this provides for greater safety of the vaccines and the expression of a “super antigen” in the form of a long, non-processed polypeptide. Sirko et al. further state that the encoding region of the HA is modified in such a way that protein production in bird cells achieves maximal yield. The main modification is codon optimisation for chicken and deletion of the proteolysis sensitive region of HA.
WO 2013/044390 describes a method of producing a virus like particle (VLP) in a plant with modified hemagglutinin (HA) wherein the modified HA protein comprises a modified proteolytic loop. The modified HA is expressed in the presence of the regulatory region Cowpea mosaic virus (CPMV) HT and the geminivirus amplification element from Bean Yellow Dwarf Virus (BeYDV).
US 2008/0069821 by Yang et al. discloses polypeptides and polynucleotides variants of influenza HA for use in the production of influenza viruses as vaccines. Reassortant influenza viruses are obtained by introducing a subset of vectors corresponding to genomic segments of a master influenza virus, in combination with complementary segments derived from the variants of influenza HA. Typically, the master strains are selected on the basis of desirable properties relevant to vaccine administration. For example, for vaccine production, e.g., for production of a live attenuated vaccine, the master donor virus strain may be selected for an attenuated phenotype, cold adaptation and/or temperature sensitivity.